Sequalprep 96 well plate kit

DNA was extracted from the samples using the Qiagen MagAttract PowerSoil DNA KF Kit (Qiagen, Hilden, Germany) optimized for the ThermoFisher KingFisher robot (Thermo Fisher Scientific, Waltham, MA). Both negative controls and positive controls were used. The positive controlled consisted of cloned Thioglobaceae SUP05, a Gamma-Proteobacteria; while negative control was extraction control negative, template-free DNA. The kit used magnetic beads to capture DNA while excluding organic inhibitors specifically. Qubit (Thermo Fisher Scientific) was used for DNA quantification and quality check. The microbial composition of samples was characterized by sequencing the V4 hypervariable region of the 16S rRNA gene using the Illumina MiSeq sequencer (Illumina, San Diego, CA) (Meisel et al., 2016 (link)). Samples that failed PCR or had spurious bands were re-amplified by optimizing PCR conditions. We used a high-throughput SequalPrep96-well plate kit (Thermo Fisher Scientific) to clean up and normalize PCR reactions.

Samples were prepared for Illumina Sequencing by 16S ribosomal RNA (rRNA) gene amplification of the bacterial community. The DNA was amplified for the hypervariable V3-V4 region with specific primers and further reamplified in a limited-cycle PCR reaction to add sequencing adapters and dual indexes. First PCR reactions were performed for each sample using KAPA HiFi HotStart PCR Kit according to manufacturer suggestions, 0.3 μM of each PCR primer: forward primer Bakt_341F 5′–CCTACGGGNGGCWGCAG-3′ and reverse primer Bakt_805R 5′–GACTACHVGGGTATCTAATCC-3′ [14 (link),15 (link)] and 12.5 ng of template DNA in a total volume of 25 μL. The PCR conditions involved a 3 min denaturation at 95 °C, followed by 25 cycles of 98 °C for 20 s, 55 °C for 30 s and 72 °C for 30 s, and a final extension at 72 °C for 5 min. Second PCR reactions added indexes and sequencing adapters to both ends of the amplified target region according to the manufacturer’s recommendations [16 ]. Negative PCR controls were included for all amplification procedures. PCR products were then one-step purified and normalized using SequalPrep 96-well plate kit (Thermo Fisher Scientific, Waltham, MA, USA) [17 (link)], pooled and pair-end sequenced in the Illumina MiSeq^® sequencer with the V3 chemistry, according to the manufacturer’s instructions (Illumina, San Diego, CA, USA) at Genoinseq (Cantanhede, Portugal).